LPS O-antigen polysaccharide length impacts outer membrane permeability of enteric gram-negative bacteria

The Gram-negative outer membrane (OM) forms the bacterial cell surface and acts as a barrier against antibiotic influx. In enteric species, the OM is covered by lipopolysaccharides (LPS) decorated with varying lengths of O-antigen (O-Ag) polysaccharide that protect bacteria against mammalian host defenses. Studies of lab-adapted Escherichia coli K-12 strains have proven instrumental in unravelling the essential processes of LPS synthesis, transport, and assembly into the OM. However, O-Ag synthesis was inactivated in K-12 strains during their lab adaption and these cells produce a non-native, truncated LPS form. Surprisingly, we found that re-activating O-Ag synthesis in K-12 permeabilizes the OM to diverse antibiotics, causing susceptibility. The O-Ag that modifies LPS is directly responsible for the compromised OM barrier. Lengthening the O-Ag polysaccharide worsens antibiotic sensitivity; while shortening it, or removing it entirely, improves antibiotic resistance in both E. coli and a closely related human pathogen, Shigella flexneri. Bacteria ensure integrity of their OM antibiotic barrier by maintaining a balanced production of long and short LPS forms, and this balance is dysfunctional in model E. coli strains. Our data reveal that long O-Ag polysaccharides are a double-edged sword: while well-recognized as critical for protection against external host assaults, their transport and assembly onto the surface comes at the inherent price of compromising the OM barrier. Our findings suggest that a balance of LPS lengths is maintained that balances host defense and OM integrity. Moreover, we identify an inherent advantage for species that produce O-Ag-lacking lipooligosaccharide (LOS) rather than LPS.